FIG 4.

Characterization of the roles of the dual cre sites in CcpA autoregulation. (A) The proposed dual-cre form for CcpA autoregulation. (B) Characteristics of the promoter region of ccpA. The transcriptional start site A is highlighted in red. The −10 and −35 regions are underlined. The CcpA-binding site cre_P is highlighted in blue. (C) Role of the cre_P site in controlling CcpA expression. cre_P, original promoter sequence of ccpA; cre_P-m, derived promoter sequence of ccpA that was mutated in the cre_P site; 824, C. acetobutylicum wild-type strain; ΔccpA, C. acetobutylicum strain with ccpA inactivation. lacZ was used here as the reporter gene, and the gray circle represents the original and mutated CcpA-binding sites, cre_P and cre_P-m. P_ccpA, promoter of the ccpA gene. (D) Role of the cre_ORF site in controlling CcpA expression. cre_ORF, ccpA coding region; cre_ORF-m, derived ccpA coding region that was mutated in the cre_ORF site. The gray circle represents the original and mutated CcpA-binding sites, cre_ORF and cre_ORF-m. The ccpA and lacZ genes were coexpressed to form a fusion protein. (E) Comparison of the influences of the cre_P and cre_ORF sites on CcpA expression. P(wt) and P(m), wild-type and cre_P mutant promoter region sequences of ccpA, respectively. ccpA(wt) and ccpA(m), wild-type and cre_ORF mutant ccpA genes, respectively. All data from the in vivo assay represent averages from two independent samples.